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Re: [ccp4bb] finicky protein |
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CCP4bb navigationCCP4bb <-- 2008 <-- March 2008 <-- 05 March 2008Subject: Re: finicky protein From: Juergen Bosch jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU Date: 2008-03-05 step - depending on your proteein (or even during expression, e.g. metals come to mind). Juergen price@UCHICAGO.EDU wrote: >Just beware that changing how you break the cells open can >change the average size of chromosome chunks, which can >change how DNA binding proteins behave in the lysate. > > >---- Original message ---- > > >>Date: Mon, 3 Mar 2008 15:21:15 +0000 >>From: Mads Gabrielsen >>Subject: [ccp4bb] finicky protein >>To: CCP4BB@JISCMAIL.AC.UK >> >>I am not a big fan of sonication. Try changing your way of >> >> >disrupting the > > >>cells. >> >>I have compared sonication vs mechanical stress on several >> >> >unrelated proteins, > > >>and for me a good old french press wins every time. If you >> >> >want to get all > > >>modern and fancy, a cell disruptor gives similar results. >> >>Cheers, >> >>Mads Gabrielsen >> >> >>[Hide Quoted Text] >> >>On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: >> >>Hi all >> >>sorry, for offtopic query... >> >>I am trying to purify my protein by Ni-NTA affinity >> >> >chromatography. After > > >>sonication as i centrifuge bacterial lysate, soon after 10 >> >> >min whole > > >>lysates >>get precipitated during loading on the column and some time >> >> >it remain > > >>soluble too. if i get purified through the column without >> >> >precipitation, > > >>it >>gets precipitated during dialysis. >>I have tried lot, by chnaging buffers, increasing salt or >> >> >deacreasing salt > > >>or no salt at are helpless. >>I do purifiaction in cold room. >> >>can any one suggest some solution? >> >>Thanks in advance. >> >>NSH >> >> >>-- >>Dr. Mads Gabrielsen >> >>GBRC, B217 >>Division of Biochemistry and Molecular Biology >>IBLS >>University of Glasgow Phone Office: 01413308119 >>G12 8QQ Phone Lab: 01413306449 >>UK E-mail: >> >> >m.gabrielsen@bio.gla.ac.uk > > >>------------------------------------------------------------ >> >> >---- > > >>This message was sent using IMP, the Internet Messaging >> >> >Program. > > > -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch CCP4bb navigationCCP4bb <-- 2008 <-- March 2008 <-- 05 March 2008 |
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